Mu-1, member of the cytokine receptor family

ABSTRACT

Polynucleotides encoding the MU-1 hematopoietin receptor superfamily chain and fragments thereof are disclosed. MU-1 proteins, antibodies (and fragments thereof) that specifically bind to MU-1 proteins (and fragments thereof), and methods for their production are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 11/957,891, filed Dec. 17, 2007, entitled “MU-1,MEMBER OF THE CYTOKINE RECEPTOR FAMILY,” which is a continuation of U.S.patent application Ser. No. 09/569,384, filed May 11, 2000, nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 09/040,005, filed Mar. 17, 1998, now U.S. Pat. No. 6,057,128,and U.S. patent application Ser. No. 09/560,766, filed Apr. 28, 2000,now abandoned. The contents of the above-referenced patent applicationsare incorporated by reference herein in their entireties.

INCORPORATION OF SEQUENCE LISTING

The Sequence Listing filed on Jan. 28, 2010, created on Jan. 28, 2010,named 019970431004ST25.TXT, having a size in bytes of 39 kb, is herebyincorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to new members of the mammalian cytokinereceptor family of proteins (including without limitation human andmurine receptor proteins), fragments thereof and recombinantpolynucleotides and cells useful for expressing such proteins.

A variety of regulatory molecules, known as hematopoietins, have beenidentified which are involved in the development and proliferation ofthe various populations of hematopoietic or blood cells. Mosthematopoietins exhibit certain biological activities by interacting witha receptor on the surface of target cells. Cytokine receptors arecommonly composed of one, two or three chains. Many cytokine receptorsand some cytokines, such as IL-12 p40, are members of the hematopoietinreceptor superfamily of proteins. Identification of new members of thehematopoietin receptor superfamily can be useful in regulation ofhematopoiesis, in regulation of immune responses and in identificationof other members of the hematopoietin superfamily, including cytokinesand receptors.

It would be desirable to identify and determine the DNA and proteinsequence for heretofore unknown members of the hematopoietin receptorsuperfamily.

SUMMARY OF THE INVENTION

In accordance with the present invention, polynucleotides encoding theMU-1 hematopoietin receptor superfamily chain are disclosed, includingwithout limitation those from murine and human sources.

In certain embodiments, the invention provides an isolatedpolynucleotide comprising a nucleotide sequence selected from the groupconsisting of: (a) the nucleotide sequence of SEQ ID NO:1; (b) thenucleotide sequence of SEQ ID NO:1 from nucleotide 236 to nucleotide1852; (c) the nucleotide sequence of SEQ ID NO:1 from nucleotide 299 tonucleotide 1852; (d) the nucleotide sequence of SEQ ID NO:1 fromnucleotide 299 to nucleotide 943; (e) a nucleotide sequence varying fromthe sequence of the nucleotide sequence specified in any of (a)-(d) as aresult of degeneracy of the genetic code; (f) a nucleotide sequencecapable of hybridizing under stringent conditions to the nucleotidespecified in any of (a)-(d); (g) a nucleotide sequence encoding aspecies homologue of the sequence of SEQ ID NO:2; and (h) an allelicvariant of the nucleotide sequence specified in any of (a)-(d).Preferably, the nucleotide sequence encodes a protein having abiological activity of the MU-1 hematopoietin receptor superfamilychain. The nucleotide sequence may be operably linked to an expressioncontrol sequence.

The invention also provides isolated polynucleotides comprising anucleotide sequence encoding a peptide or protein comprising an aminoacid sequence selected from the group consisting of: (a) the amino acidsequence of SEQ ID NO:2; (b) the amino acid sequence of SEQ ID NO:2 fromamino acids 22 to 538; (c) the amino acid sequence of SEQ ID NO:2 fromamino acids 22 to 236; (d) the amino acid sequence of SEQ ID NO:2 fromamino acids 1 to 236; and (e) fragments of (a)-(d) having a biologicalactivity of the MU-1 hematopoietin receptor superfamily chain.

In another embodiment, the invention provides an isolated polynucleotidecomprising a nucleotide sequence selected from the group consisting of:(a) the nucleotide sequence of SEQ ID NO:9; (b) a nucleotide sequencevarying from the sequence of the nucleotide sequence specified in (a) asa result of degeneracy of the genetic code; (c) a nucleotide sequencecapable of hybridizing under stringent conditions to the nucleotidesequence specified in (a); and (d) an allelic variant of the nucleotidesequence specified in (a).

The invention also provides isolated polynucleotides comprising anucleotide sequence encoding a peptide or protein comprising an aminoacid sequence selected from the group consisting of: (a) the amino acidsequence of SEQ ID NO:10; and (b) fragments of (a) having a biologicalactivity of the MU-1 hematopoietin receptor superfamily chain.

Host cells, preferably mammalian cells, transformed with thepolynucleotides are also provided.

In other embodiments, the invention provides a process for producing aMU-1 protein. The process comprises: (a) growing a culture of the hostcell of the present invention in a suitable culture medium; and (b)purifying the human MU-1 protein from the culture. Proteins producedaccording to these methods are also provided.

The present invention also provides for an isolated MU-1 proteincomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:2; (b) the amino acid sequenceof SEQ ID NO:2 from amino acids 22 to 538; (c) the amino acid sequenceof SEQ ID NO:2 from amino acids 22 to 236; (d) the amino acid sequenceof SEQ ID NO:2 from amino acids 1 to 236; and (e) fragments of (a)-(d)having a biological activity of the MU-1 hematopoietin receptorsuperfamily chain.

The present invention also provides for an isolated MU-1 proteincomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:10; and (b) fragments of (a)having a biological activity of the MU-1 hematopoietin receptorsuperfamily chain.

Murine MU-1 (SEQ ID NO:10) has 65% identity with human MU-1.

In other preferred embodiments, the specified amino acid sequence ispart of a fusion protein (with an additional amino acid sequence notderived from MU-1). Preferred fusion proteins comprise an antibodyfragment, such as an Fc fragment.

Pharmaceutical compositions comprising a protein of the presentinvention and a pharmaceutically acceptable carrier are also provided.

The present invention further provides for compositions comprising anantibody that specifically reacts with a protein of the presentinvention.

In one embodiment, a MU-1 nucleic acid molecule of the invention is atleast 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the nucleotidesequence (e.g., to the entire length of the nucleotide sequence) shownin SEQ ID NO:1 or 9.

In another preferred embodiment, the nucleic acid molecule consists ofthe nucleotide sequence shown in SEQ ID NO:1 or 9. In another preferredembodiment, the nucleic acid molecule includes a fragment of at least50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, or more nucleotides (e.g., contiguousnucleotides) of the nucleotide sequence of SEQ ID NO:1 or 9, or acomplement thereof.

In a preferred embodiment, the MU-1 protein family member has an aminoacid sequence at least about 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or moreidentical to the amino acid sequence of SEQ ID NO:2 or 10.

In another embodiment, the invention features fragments of the proteinhaving the amino acid sequence of SEQ ID NO:2 or 10, wherein thefragment comprises at least 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100amino acids (e.g., contiguous amino acids) of the amino acid sequence ofSEQ ID NO:2 or 10.

In another aspect, the present invention provides a method for detectingthe presence of a MU-1 nucleic acid molecule, protein or polypeptide ina biological sample by contacting the biological sample with an agentcapable of detecting a MU-1 nucleic acid molecule, protein orpolypeptide such that the presence of a MU-1 nucleic acid molecule,protein or polypeptide is detected in the biological sample.

In another aspect, the present invention provides a method for detectingthe presence of MU-1 activity in a biological sample by contacting thebiological sample with an agent capable of detecting an indicator ofMU-1 activity such that the presence of MU-1 activity is detected in thebiological sample.

In another aspect, the invention provides a method for modulating MU-1activity comprising contacting a cell capable of expressing MU-1 with anagent that modulates MU-1 activity such that MU-1 activity in the cellis modulated. In one embodiment, the agent inhibits MU-1 activity. Inanother embodiment, the agent stimulates MU-1 activity. In oneembodiment, the agent is an antibody that specifically binds to a MU-1protein. In another embodiment, the agent modulates expression of MU-1by modulating transcription of a MU-1 gene or translation of a MU-1mRNA. In yet another embodiment, the agent is a nucleic acid moleculehaving a nucleotide sequence that is antisense to the coding strand of aMU-1 mRNA or a MU-1 gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant or unwantedMU-1 protein or nucleic acid expression or activity (e.g., an MU-1associated disorder) by administering an agent that is a MU-1 modulatorto the subject. In one embodiment, the MU-1 modulator is an MU-1protein. In another embodiment, the MU-1 modulator is an MU-1 nucleicacid molecule. In yet another embodiment, the MU-1 modulator is apeptide, peptidomimetic, or other small molecule.

The present invention also provides diagnostic assays for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding aMU-1 protein; (ii) misregulation of the MU-1 gene; and (iii) aberrantpost-translational modification of an MU-1 protein, wherein a wild-typeform of the gene encodes a protein with a MU-1 activity.

In another aspect the invention provides methods for identifying acompound that binds to or modulates the activity of a MU-1 protein, byproviding an indicator composition comprising a MU-1 protein having MU-1activity, contacting the indicator composition with a test compound, anddetermining the effect of the test compound on MU-1 activity (e.g., STATphosphorylation, e.g., STAT 3 or STAT 5 phosphorylation) in theindicator composition to identify a compound that modulates the activityof a MU-1 protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the full-length cDNA sequence of murine MU-1. Thenucleotide sequence corresponds to nucleotides 1-2628 of SEQ ID NO:9.FIG. 1A depicts the cDNA sequence of murine MU-1 from nucleotide 1 to1450, and FIG. 1B depicts the cDNA sequence of murine MU-1 fromnucleotide 1451 to 2628.

FIG. 2 depicts the amino acid sequence of murine MU-1 (corresponding tothe amino acids 1-529 of SEQ ID NO:10). There is a predicted leadersequence at amino acids 1-19, which was predicted by SPScan with a scoreof 10.1 (bold-face type). There is a predicted transmembrane domain atamino acids 237-253 (underlined). Predicted signaling motifs include thefollowing regions: Box 1: amino acids 265-274, and Box 2: amino acids311-324 (bold and underlined); six tyrosines are located at amino acidpositions 281, 319, 361, 369, 397, and 510. The WSXWS motif (SEQ IDNO:8) is located at amino acid residue 214 to amino acid residue 218 (inlarge, bold-face type). Potential STAT docking sites include amino acids393-398 and amino acids 510-513.

FIG. 3 depicts the GAP comparison of human and murine MU-1 cDNAsequences (corresponding to nucleic acids 1-1909, 1960-2050, and2151-2665 of SEQ ID NO:1 for the purposes of an alignment, and nucleicacids 151-2628 of SEQ ID NO:9, respectively). HuMU-1=human MU-1,murMU-1=murine MU-1. Gap Parameters: Gap Weight=50, AverageMatch=10.000, Length Weight=3, Average Mismatch=0.000. PercentIdentity=66.116. FIG. 3A is a comparison of nucleotides 1-721 of humanMU-1 of SEQ ID NO:1 to nucleotides 151-892 of mouse MU-1; FIG. 3B is acomparison of nucleotides 722-1509 of human MU-1 of SEQ ID NO:1 tonucleotides 893-1674 of mouse MU-1; FIG. 3C is a comparison ofnucleotides 1510-2404 of human MU-1 of SEQ ID NO:1, wherein nucleicacids 1910-1959 and 2051-2150 of SEQ ID NO:1 have been removed for thepurposes of the alignment, to nucleotides 1675-2368 of mouse MU-1; andFIG. 3D is a comparison of nucleotides 2405-2665 of human MU-1 of SEQ IDNO:1 to nucleotides 2369-2628 of mouse MU-1.

FIG. 4 depicts a GAP comparison of the human MU-1 protein (correspondingto amino acids 1-538 of SEQ ID NO:2) and the murine MU-1 protein(corresponding to amino acids 1-529 of SEQ ID NO:10): BLOSUM62 aminoacid substitution matrix (Henikoff and Henikoff (1992) “Amino acidsubstitution matrices from protein blocks” Proc. Natl. Acad. Sci. USA89:10915-19). Gap parameters=Gap Weight: 8, Average Match=2.912, LengthWeight=2, Average Mismatch=−2.003. Percent Identity=65.267.

FIG. 5 depicts a multiple sequence alignment of the amino acids of humanMU-1 (corresponding to SEQ ID NO:2), murine MU-1 (corresponding to SEQID NO:10), and human IL-2 beta chain (GenBank Accession No. AAA59143.1,corresponding to SEQ ID NO:17). Leader and transmembrane domains areunderlined. Conserved cytokine receptor module motifs are indicated bybold-face type. Potential signaling regions are indicated by underliningand bold-face type.

FIG. 6 depicts signaling through MU-1. MU-1 phosphorylates STAT 5 inClone E7 EPO-MU-1 chimera. Under the conditions specified in Example 3,signaling through MU-1 results in the phosphorylation of STAT 5 at alltime points tested. Treatment of controls or the chimeric BAF-3 cellswith IL-3 resulted in phosphorylation of STAT 3, but not STAT 1 or STAT5.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present application have for the first timeidentified and provided polynucleotides encoding the MU-1 hematopoietinreceptor superfamily chain (hereinafter “MU-1” or “MU-1 protein”),including without limitation polynucleotides encoding human and murineMU-1.

In a particularly preferred embodiment, the MU-1 protein and nucleicacid molecules of the present invention are human MU-1 molecules. A70-amino acid region of the human IL-5 receptor(LMTNAFISIIDDLSKYDVQVRAAVSSMCREAGLWSEWSQPIYVGNDEHKPLREWFVIVIMATICFILLIL, SEQ ID NO:3) was used tosearch the GenBank EST database using the TBLASTN algorithm. A sequencewithin the genomic BAC clone AC002303 from human chromosome 16p12 wasidentified with homology to this region, suggesting that this segmentmight encode a gene for a novel hematopoietin receptor. Examination ofopen reading frames within 1000 by of nucleotide 40,886 revealed a 270by open frame which when used in a BLASTP search of GenPept exclusivelyidentified members of the cytokine receptor family. A stop codon presentat the end of this reading frame was interpreted as an indication oftransition over an exon/intron border.

It was then determined whether RNA was transcribed from a gene containedwithin this BAC clone from chromosome 16p12. PCR primers weresynthesized based on the largest ORF segment which contained peptidesequence conserved within the cytokine receptor family. PrimersGAGTCCGAGGAGAAAGCTGATCTCA (5p) (SEQ ID NO:4) andGAAAGATGACCGGGTCACTCCATT (3p) (SEQ ID NO:5) were used in PCRs to screenphage libraries from various human tissues (Clontech). PCR products ofthe expected 164 by size that specifically hybridized to a ³²P-labeledoligonucleotide of the sequence ACTCGAGCTATGAGCTGCAGGTGC GGGCA (SEQ IDNO:6) were observed in phage from lung, kidney, placenta and heart.Using the oligonucleotide ACTCGAGCTATGAGCTGCAGGT GCGGGCA (SEQ ID NO:7) afull-length cDNA clone NN14-1b (MU-1) was identified, purified, andsequenced. The DNA sequence and the predicted amino acid sequence areshown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The predicted aminoacid sequence of the human MU-1 receptor chain includes a putativesignal sequence from either amino acids 1-21 or 1-19. The mature humanMU-1 is believed to have the sequence of amino acids 22-538 or 20-538 ofSEQ ID NO:2. A transmembrane domain is found at amino acids 237-254.

In another particularly preferred embodiment, the MU-1 protein andnucleic acid molecules of the present invention are murine MU-1molecules. To identify the polynucleotide sequence encoding the murineMU-1 protein, a partial fragment of the murine homolog of the MU-1receptor was isolated by PCR from mouse cDNA using oligonucleotidesderived from the human sequence, as described in Example 1. The DNAsequence of this fragment was determined, and two oligonucleotides werederived from an internal portion of this fragment with the followingsequences:

TTGAACGTGACTGTGGCCTT (5p) (SEQ ID NO: 13) TGAATGAAGTGCCTGGCTGA (3p).(SEQ ID NO: 14)The oligonucleotides were used to amplify an internal 262 nucleotidefragment of the original PCR product (corresponding to nucleotides781-1043 of the murine cDNA sequence of FIG. 1 and SEQ ID NO:9) to useas a hybridization probe to screen a cDNA library isolated from the 2D6T cell line. DNA sequence was determined from two independent clones.Clone 6 was sequenced and confirmed to be the full-length murine homologof human MU-1.

The full-length nucleotide sequence of murine MU-1 is shown in FIG. 1(corresponding to nucleotides 1-2628 of SEQ ID NO:9). The nucleotidesequence has a predicted leader sequence at nucleotides 407-463, codingsequence at nucleotides 407-1993, and a termination codon at nucleotides1994-1996. Nucleotides 1-406 correspond to the 5′ untranslated regionand nucleotides 1997-2628 correspond to the 3′ untranslated region. Thepredicted protein sequence of murine MU-1 is shown in FIG. 2(corresponding to amino acids 1-529 of SEQ ID NO:10).

The murine MU-1 protein contains a predicted leader sequence determinedby SPScan (score=10.1) (corresponding to amino acids 1-19 of SEQ IDNO:10), and a predicted transmembrane domain (corresponding to aminoacids 237-253 of SEQ ID NO:10). Predicted signaling motifs include thefollowing regions: Box 1: amino acids 265-274 of SEQ ID NO:10, Box 2:amino acids 311-324 of SEQ ID NO:10, six tyrosine residues at positions281, 319, 361, 369, 397, and 510 of SEQ ID NO:10. Potential STAT dockingsites include but are not limited to: STAT 5: EDDGYPA (corresponding toSEQ ID NO:18); STAT 3: YLQR (corresponding to SEQ ID NO:19).

The open reading frame of MU-1 encodes a novel member of thehematopoietin receptor family. In a preferred embodiment, MU-1 has aleader sequence, conserved cysteine pairs, PP, and WSXWS (SEQ ID NO:8)motifs characteristic of the family, as well as a transmembrane domainand an extensive cytoplasmic domain. MU-1 also contains a conserved PXPPas well as Box 1 and Box 2 signaling motifs in the cytoplasmic domain(see FIG. 5). These domains are conserved between murine MU-1 and humanMU-1. Subsequent FASTA alignment of MU-1 sequence with GenPept showedgreatest homology with human IL-2Rb (see FIG. 5).

The human MU-1 cDNA was deposited with the American Type CultureCollection on March 10, 1998, as accession number ATCC 98687.

By Northern analysis, as described in Example 4, murine MU-1 wasdetected in adult murine spleen, lung, and heart tissues. Human MU-1 wasdetected in adult human lymphoid tissues, PBLs, thymus, spleen and lymphnode, and in fetal lung.

Any forms of MU-1 proteins of less than full length are encompassedwithin the present invention and are referred to herein collectivelywith full-length and mature forms as “MU-1” or “MU-1 proteins.” MU-1proteins of less than full length may be produced by expressing acorresponding fragment of the polynucleotide encoding the full-lengthMU-1 protein (SEQ ID NO:2 or SEQ ID NO:10). These correspondingpolynucleotide fragments are also part of the present invention.Modified polynucleotides as described above may be made by standardmolecular biology techniques, including construction of appropriatedesired deletion mutants, site-directed mutagenesis methods or by thepolymerase chain reaction using appropriate oligonucleotide primers.

For the purposes of the present invention, a protein has “a biologicalactivity of the MU-1 hematopoietin receptor superfamily chain” if itpossesses one or more of the biological activities of the correspondingmature MU-1 protein. In one embodiment, MU-1 activity includesinteraction with STAT molecules (e.g., STAT 5, STAT 3). In anotherembodiment, the MU-1 protein activity does not bind to any knowncytokines.

MU-1 or active fragments thereof (MU-1 proteins) may be fused to carriermolecules such as immunoglobulins or immunoglobulin fragments. Forexample, soluble forms of the MU-1 may be fused through “linker”sequences to the Fc portion of an immunoglobulin. Other fusion proteins,such as those with GST, Lex-A or MBP, may also be used.

The invention also encompasses allelic variants of the nucleotidesequences as set forth in SEQ ID NO:1 and SEQ ID NO:9, that is,naturally occurring alternative forms of the isolated polynucleotide ofSEQ ID NO:1 or SEQ ID NO:9 that also encode MU-1 proteins, preferablythose proteins having a biological activity of MU-1. Also included inthe invention are isolated polynucleotides that hybridize to thenucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:9 under highlystringent conditions (for example, 0.1×SSC at 65° C.). Isolatedpolynucleotides which encode MU-1 proteins but which differ from thenucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:9 by virtue ofthe degeneracy of the genetic code are also encompassed by the presentinvention. Variations in the nucleotide sequence as set forth in SEQ IDNO:1 or SEQ ID NO:9 that are caused by point mutations or by inducedmodifications are also included in the invention.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% identical to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 70%, more preferably at leastabout 80%, even more preferably at least about 85% or 90% identical toeach other typically remain hybridized to each other. Such stringentconditions are known to those skilled in the art and can be found inCurrent Protocols in Molecular Biology, John Wiley & Sons, NY (1989),6.3.1-6.3.6. A preferred, nonlimiting example of stringent hybridizationconditions is hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at50° C., preferably at 55° C., more preferably at 60° C., and even morepreferably at 65° C. Ranges that span the above recited values, e.g.,55-60° C., or 50-65° C. are encompassed by the present invention.Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1 or9 corresponds to a naturally occurring nucleic acid molecule. As usedherein, a “naturally occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein).

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnonidentical sequences can be disregarded for comparison purposes). In apreferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-53) algorithm which has been incorporatedinto the GAP program in GCG software package, using either a Blosum 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in GCG software package, using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percentidentity between two amino acid or nucleotide sequences is determinedusing the algorithm of Myers and Miller ((1988) Comput. Appl. Biosci.4:11-17), which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to MU-1 nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=100,wordlength=3 to obtain amino acid sequences homologous to MU-1 proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25(17):3389-402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used (see, The National Center forBiotechnology Information website).

The present invention also provides polynucleotides encoding homologuesof the human MU-1 from other animal species, particularly othermammalian species. Species homologues can be identified and isolated bymaking probes or primers from the murine or human sequences disclosedherein and screening a library from an appropriate species, such as forexample libraries constructed from PBMCs, thymus or testis of therelevant species.

The isolated polynucleotides of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al. (1991) Nuc. Acids Res. 19:4485-90,in order to produce the MU-1 protein recombinantly. Many suitableexpression control sequences are known in the art. General methods ofexpressing recombinant proteins are also known and are exemplified inKaufman (1990) Meth. Enzymol. 185:537-66. As defined herein, “operablylinked” means enzymatically or chemically ligated to form a covalentbond between the isolated polynucleotide of the invention and theexpression control sequence, in such a way that the MU-1 protein isexpressed by a host cell that has been transformed (transfected) withthe ligated polynucleotide/expression control sequence.

A number of types of cells may act as suitable host cells for expressionof the MU-1 protein. Any cell type capable of expressing functional MU-1protein may be used. Suitable mammalian host cells include, for example,monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1cells, other transformed primate cell lines, normal diploid cells, cellstrains derived from in vitro culture of primary tissue, primaryexplants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK, Rat2, BaF3,32D, FDCP-1, PC12, M1x or C2C12 cells.

The MU-1 protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif. (the MaxBac® kit), and such methods are well known inthe art, as described in Summers and Smith (1987) Texas AgriculturalExperiment Station Bulletin No. 1555, incorporated herein by reference.Soluble forms of the MU-1 protein may also be produced in insect cellsusing appropriate isolated polynucleotides as described above.

Alternatively, the MU-1 protein may be produced in lower eukaryotes suchas yeast or in prokaryotes such as bacteria. Suitable yeast strainsinclude Saccharomyces cerevisiae, Schizosaccharomyces pombe,Kluyveromyces strains, Candida, or any yeast strain capable ofexpressing heterologous proteins. Suitable bacterial strains includeEscherichia coli, Bacillus subtilis, Salmonella typhimurium, or anybacterial strain capable of expressing heterologous proteins.

Expression in bacteria may result in formation of inclusion bodiesincorporating the recombinant protein. Thus, refolding of therecombinant protein may be required in order to produce active or moreactive material. Several methods for obtaining correctly foldedheterologous proteins from bacterial inclusion bodies are known in theart. These methods generally involve solubilizing the protein from theinclusion bodies, then denaturing the protein completely using achaotropic agent. When cysteine residues are present in the primaryamino acid sequence of the protein, it is often necessary to accomplishthe refolding in an environment that allows correct formation ofdisulfide bonds (a redox system). General methods of refolding aredisclosed in Kohno (1990) Meth. Enzym. 185:187-95. EP 0433225 and U.S.Pat. No. 5,399,677 describe other appropriate methods.

The MU-1 protein of the invention may also be expressed as a product oftransgenic animals, e.g., as a component of the milk of transgenic cows,goats, pigs, or sheep which are characterized by somatic or germ cellscontaining a polynucleotide sequence encoding the MU-1 protein.

The MU-1 protein of the invention may be prepared by growing a cultureof transformed host cells under culture conditions necessary to expressthe desired protein. The resulting expressed protein may then bepurified from the culture medium or cell extracts. Soluble forms of theMU-1 protein of the invention can be purified from conditioned media.Membrane-bound forms of MU-1 protein of the invention can be purified bypreparing a total membrane fraction from the expressing cell andextracting the membranes with a nonionic detergent such as Triton X-100.

The MU-1 protein can be purified using methods known to those skilled inthe art. For example, the MU-1 protein of the invention can beconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) or polyetheyleneimine(PEI) groups. The matrices can be acrylamide, agarose, dextran,cellulose or other types commonly employed in protein purification.Alternatively, a cation exchange step can be employed. Suitable cationexchangers include various insoluble matrices comprising sulfopropyl orcarboxymethyl groups. Sulfopropyl groups are preferred (e.g.,S-Sepharose® columns). The purification of the MU-1 protein from culturesupernatant may also include one or more column steps over such affinityresins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue3GA Sepharose®; or by hydrophobic interaction chromatography using suchresins as phenyl ether, butyl ether, or propyl ether; or byimmunoaffinity chromatography. Finally, one or more reverse-phase highperformance liquid chromatography (RP-HPLC) steps employing hydrophobicRP-HPLC media, e.g., silica gel having pendant methyl or other aliphaticgroups, can be employed to further purify the MU-1 protein. Affinitycolumns including antibodies to the MU-1 protein can also be used inpurification in accordance with known methods. Some or all of theforegoing purification steps, in various combinations or with otherknown methods, can also be employed to provide a substantially purifiedisolated recombinant protein. Preferably, the isolated MU-1 protein ispurified so that it is substantially free of other mammalian proteins.

MU-1 proteins of the invention may also be used to screen for agentsthat are capable of binding to MU-1. Binding assays using a desiredbinding protein, immobilized or not, are well known in the art and maybe used for this purpose using the MU-1 protein of the invention.Purified cell based or protein based (cell free) screening assays may beused to identify such agents. For example, MU-1 protein may beimmobilized in purified form on a carrier and binding or potentialligands to purified MU-1 protein may be measured.

MU-1 proteins, purified from cells or recombinantly produced, may beused as a pharmaceutical composition when combined with apharmaceutically acceptable carrier. Such a composition may contain, inaddition to MU-1 or inhibitor and carrier, various diluents, fillers,salts, buffers, stabilizers, solubilizers, and other materials wellknown in the art. The term “pharmaceutically acceptable” means anontoxic material that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration.

The pharmaceutical composition of the invention may also containcytokines, lymphokines, or other hematopoietic factors such as M-CSF,GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-14, IL-15, G-CSF, stem cell factor, and erythropoietin.The pharmaceutical composition may also include anti-cytokineantibodies. The pharmaceutical composition may contain thrombolytic oranti-thrombotic factors such as plasminogen activator and Factor VIII.The pharmaceutical composition may further contain otheranti-inflammatory agents. Such additional factors and/or agents may beincluded in the pharmaceutical composition to produce a synergisticeffect with isolated MU-1 protein, or to minimize side effects caused bythe isolated MU-1 protein. Conversely, isolated MU-1 protein may beincluded in formulations of the particular cytokine, lymphokine, otherhematopoietic factor, thrombolytic or anti-thrombotic factor, oranti-inflammatory agent to minimize side effects of the cytokine,lymphokine, other hematopoietic factor, thrombolytic or anti-thromboticfactor, or anti-inflammatory agent.

The pharmaceutical composition of the invention may be in the form of aliposome. In the liposome, isolated MU-1 protein and pharmaceuticallyacceptable carriers are combined with amphipathic agents, such as lipidsthat exist in aggregated form, such as micelles, insoluble monolayers,liquid crystals, or lamellar layers, in aqueous solution. Suitablelipids for liposomal formulation include, without limitation,monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,saponin, bile acids, and the like. Preparation of such liposomalformulations is within the level of skill in the art, as disclosed, forexample, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and4,737,323, all of which are incorporated herein by reference.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, e.g.,amelioration of symptoms of, healing of, or increase in rate of healingof such conditions. When applied to an individual active ingredient,administered alone, the term refers to that ingredient alone. Whenapplied to a combination, the term refers to combined amounts of theactive ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of isolated MU-1 protein isadministered to a mammal. Isolated MU-1 protein may be administered inaccordance with the method of the invention either alone or incombination with other therapies such as treatments employing cytokines,lymphokines or other hematopoietic factors. When coadministered with oneor more cytokines, lymphokines or other hematopoietic factors, MU-1protein may be administered either simultaneously with the cytokine(s),lymphokine(s), other hematopoietic factor(s), thrombolytic oranti-thrombotic factors, or sequentially. If administered sequentially,the attending physician will decide on the appropriate sequence ofadministering MU-1 protein in combination with cytokine(s),lymphokine(s), other hematopoietic factor(s), thrombolytic oranti-thrombotic factors.

Administration of MU-1 protein used in the pharmaceutical composition orto practice the method of the present invention can be carried out in avariety of conventional ways, such as oral ingestion, inhalation, orcutaneous, subcutaneous, or intravenous injection. Intravenousadministration to the patient is preferred.

When a therapeutically effective amount of MU-1 protein is administeredorally, MU-1 protein will be in the form of a tablet, capsule, powder,solution or elixir. When administered in tablet form, the pharmaceuticalcomposition of the invention may additionally contain a solid carriersuch as a gelatin or an adjuvant. The tablet, capsule, and powdercontain from about 5 to 95% MU-1 protein, and preferably from about 25to 90% MU-1 protein. When administered in liquid form, a liquid carriersuch as water, petroleum, oils of animal or plant origin such as peanutoil, mineral oil, soybean oil, or sesame oil, or synthetic oils may beadded. The liquid form of the pharmaceutical composition may furthercontain physiological saline solution, dextrose or other saccharidesolution, or glycols such as ethylene glycol, propylene glycol orpolyethylene glycol. When administered in liquid form, thepharmaceutical composition contains from about 0.5 to 90% by weight ofMU-1 protein, and preferably from about 1 to 50% MU-1 protein.

When a therapeutically effective amount of MU-1 protein is administeredby intravenous, cutaneous or subcutaneous injection, MU-1 protein willbe in the form of a pyrogen-free, parenterally acceptable aqueoussolution. The preparation of such parenterally acceptable proteinsolutions, having due regard to pH, isotonicity, stability, and thelike, is within the skill in the art. A preferred pharmaceuticalcomposition for intravenous, cutaneous, or subcutaneous injection shouldcontain, in addition to MU-1 protein an isotonic vehicle such as sodiumchloride injection, Ringer's injection, dextrose injection, dextrose andsodium chloride injection, lactated Ringer's injection, or other vehicleas known in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art.

The amount of MU-1 protein in the pharmaceutical composition of thepresent invention will depend upon the nature and severity of thecondition being treated, and on the nature of prior treatments that thepatient has undergone. Ultimately, the attending physician will decidethe amount of MU-1 protein with which to treat each individual patient.Initially, the attending physician will administer low doses of MU-1protein and observe the patient's response. Larger doses of MU-1 proteinmay be administered until the optimal therapeutic effect is obtained forthe patient, and at that point the dosage is not generally increasedfurther. It is contemplated that the various pharmaceutical compositionsused to practice the method of the present invention should containabout 0.1 μg to about 100 mg of MU-1 protein per kg body weight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. It is contemplated that theduration of each application of the MU-1 protein will be in the range of12 to 24 hours of continuous intravenous administration. Ultimately theattending physician will decide on the appropriate duration ofintravenous therapy using the pharmaceutical composition of the presentinvention.

The polynucleotide and proteins of the present invention are expected toexhibit one or more of the uses or biological activities (includingthose associated with assays cited herein) identified below. Uses oractivities described for proteins of the present invention may beprovided by administration or use of such proteins or by administrationor use of polynucleotides encoding such proteins (such as, for example,in gene therapies or vectors suitable for introduction of DNA).

Cytokine and Cell Proliferation/Differentiation Activity

A protein of the present invention may exhibit cytokine, cellproliferation (either inducing or inhibiting) or cell differentiation(either inducing or inhibiting) activity or may induce production ofother cytokines in certain cell populations.

Many protein factors discovered to date, including all known cytokines,have exhibited activity in one or more factor-dependent cellproliferation assays, and hence the assays serve as a convenientconfirmation of cytokine activity. The activity of a protein of thepresent invention is evidenced by any one of a number of routinefactor-dependent cell proliferation assays for cell lines including,without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preBM+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mole and CMK.

BAF-3 cells expressing huEPOR-huMU chimeric receptors proliferate inresponse to huEPO. In order to test the capacity for the MU receptor tosignal through their cytoplasmic domain, BAF-3 cells were engineered toexpress EPOr/MU(cyto) chimeric receptors and assayed for ³H thymidineincorporation in the presence of EPO. BAF-3 cells expressing intact EPOrmolecules proliferate in response to EPO while parent BAF-3 cells donot. The A5 clone, possessing the chimeric EPOr/MU(cyto) proliferates inresponse to EPO demonstrating that the cytoplasmic portion of MU-1 cansustain a proliferative signal. The BAF-3 cells which express EPOr ontheir surface also respond to EPO.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for T cell or thymocyte proliferation include without limitationthose described in: Current Protocols in Immunology, J. E. Coligan etal. eds., Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In Vitro Assays for Mouse Lymphocyte Function 3.1-3.19;Chapter 7, Immunologic Studies in Humans); Takai et al. (1986) J.Immunol. 137:3494-500; Bertagnolli et al. (1990) J. Immunol.145:1706-12; Bertagnolli et al. (1991) Cell. Immunol. 133:327-41;Bertagnolli et al. (1992) J. Immunol. 149:3778-83; Bowman et al. (1994)J. Immunol. 152:1756-61.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Kruisbeek and Shevach (1994) “Polyclonal T cellStimulation” In Current Protocols in Immunology, J. E. Coligan et al.eds., Vol. 1, pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto; andSchreiber (1994) “Measurement of Mouse and Human Interferon ≢5” InCurrent Protocols in Immunology. J. E. Coligan et al. eds., Vol. 1, pp.6.8.1-6.8.8, John Wiley and Sons, Toronto.

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Bottomly et al. (1991) “Measurement of Human and Murine Interleukin 2and Interleukin 4” In Current Protocols in Immunology, J. E. Coligan etal. eds., Vol. 1, pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto;deVries et al. (1991) J. Exp. Med. 173:1205-11; Moreau et al. (1988)Nature 336:690-92; Greenberger et al. (1983) Proc. Natl. Acad. Sci. USA80:2931-38, 1983; Nordan (1991) “Measurement of Mouse and HumanInterleukin 6” In Current Protocols in Immunology, J. E. Coligan et al.eds., Vol. 1, pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto; Smith etal. (1986) Proc. Natl. Acad. Sci. USA 83:1857-61; Bennett et al. (1991)“Measurement of Human Interleukin 11” In Current Protocols inImmunology. J. E. Coligan et al. eds., Vol. 1, p. 6.15.1, John Wiley andSons, Toronto; Ciarletta et al. (1991) “Measurement of Mouse and HumanInterleukin 9” In Current Protocols in Immunology, J. E. Coligan et al.eds., Vol. 1, p. 6.13.1, John Wiley and Sons, Toronto.

Assays for T cell clone responses to antigens (which will identify,among others, proteins that affect APC-T cell interactions as well asdirect T cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, J. E. Coligan et al. eds., Pub. GreenePublishing Associates and Wiley-Interscience (Chapter 3, In Vitro Assaysfor Mouse Lymphocyte Function, 3.1-3.19; Chapter 6, Cytokines and theirCellular Receptors; Chapter 7, Immunologic Studies in Humans);Weinberger et al. (1980) Proc. Natl. Acad. Sci. USA 77:6091-95;Weinberger et al. (1981) Eur. J. Immunol. 11:405-11; Takai et al. (1986)J. Immunol. 137:3494-500; Takai et al. (1988) J. Immunol. 140:508-12.

Immune Stimulating or Suppressing Activity

A protein of the present invention may also exhibit immune-stimulatingor immune-suppressing activity, including without limitation theactivities for which assays are described herein. A protein may beuseful in the treatment of various immune deficiencies and disorders(including severe combined immunodeficiency (SCID)), e.g., in regulating(up or down) growth and proliferation of T and/or B lymphocytes, as wellas effecting the cytolytic activity of NK cells and other cellpopulations. These immune deficiencies may be genetic or be caused byviral (e.g., HIV) as well as bacterial or fungal infections, or mayresult from autoimmune disorders. More specifically, infectious diseasescauses by viral, bacterial, fungal or other infection may be treatableusing a protein of the present invention, including infections by HIV,hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malariaspp. and various fungal infections such as candidiasis. Of course, inthis regard, a protein of the present invention may also be useful wherea boost to the immune system generally may be desirable, e.g., in thetreatment of cancer.

Autoimmune disorders which may be treated using a protein of the presentinvention include, for example, connective tissue disease, multiplesclerosis, systemic lupus erythematosus (SLE), rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin-dependent diabetes mellitis, myasthenia gravis,graft-versus-host disease and autoimmune inflammatory eye disease. Sucha protein of the present invention may also to be useful in thetreatment of allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems. Otherconditions, in which immune suppression is desired (including, forexample, organ transplantation), may also be treatable using a proteinof the present invention.

The MU-1 DNA also maps to the chromosomal locus for Crohn's disease. Asa result, proteins of the present invention may be used to treat Crohn'sdisease and other inflammatory bowel diseases.

Using the proteins of the invention it may also be possible to regulateimmune responses, in a number of ways. Downregulation may be in the formof inhibiting or blocking an immune response already in progress or mayinvolve preventing the induction of an immune response. The functions ofactivated T cells may be inhibited by suppressing T cell responses or byinducing specific tolerance in T cells, or both. Immunosuppression of Tcell responses is generally an active, non-antigen-specific, processthat requires continuous exposure of the T cells to the suppressiveagent. Tolerance, which involves inducing nonresponsiveness or anergy inT cells, is distinguishable from immunosuppression in that it isgenerally antigen-specific and persists after exposure to the tolerizingagent has ceased. Operationally, tolerance can be demonstrated by thelack of a T cell response upon reexposure to specific antigen in theabsence of the tolerizing agent.

Downregulating or preventing one or more antigen functions (includingwithout limitation B lymphocyte antigen functions (such as, for example,B7)), e.g., preventing high level lymphokine synthesis by activated Tcells, will be useful in situations of tissue, skin and organtransplantation and in graft-versus-host disease (GVHD). For example,blockage of T cell function should result in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign by Tcells, followed by an immune reaction that destroys the transplant. Theadministration of a molecule which inhibits or blocks interaction of aB7 lymphocyte antigen with its natural ligand(s) on immune cells (suchas a soluble, monomeric form of a peptide having B7-2 activity alone orin conjunction with a monomeric form of a peptide having an activity ofanother B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody),prior to transplantation can lead to the binding of the molecule to thenatural ligand(s) on the immune cells without transmitting thecorresponding costimulatory signal. Blocking B lymphocyte antigenfunction in this matter prevents cytokine synthesis by immune cells,such as T cells, and thus acts as an immunosuppressant. Moreover, thelack of costimulation may also be sufficient to anergize the T cells,thereby inducing tolerance in a subject. Induction of long-termtolerance by B lymphocyte antigen-blocking reagents may avoid thenecessity of repeated administration of these blocking reagents. Toachieve sufficient immunosuppression or tolerance in a subject, it mayalso be necessary to block the function of a combination of B lymphocyteantigens.

The efficacy of particular blocking reagents in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. Examples of appropriate systemswhich can be used include allogeneic cardiac grafts in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA4Ig fusion proteinsin vivo as described in Lenschow et al. (1992) Science 257:789-92 andTurka et al. (1992) Proc. Natl. Acad. Sci. USA 89:11102-05. In addition,murine models of GVHD (see Paul ed. (1989) Fundamental Immunology, RavenPress, New York, pp. 846-47) can be used to determine the effect ofblocking B lymphocyte antigen function in vivo on the development ofthat disease.

Blocking antigen function may also be therapeutically useful fortreating autoimmune diseases. Many autoimmune disorders are the resultof inappropriate activation of T cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive T cells may reduce or eliminate disease symptoms.Administration of reagents that block costimulation of T cells bydisrupting receptor:ligand interactions of B lymphocyte antigens can beused to inhibit T cell activation and prevent production ofautoantibodies or T cell-derived cytokines which may be involved in thedisease process. Additionally, blocking reagents may induceantigen-specific tolerance of autoreactive T cells which could lead tolong-term relief from the disease. The efficacy of blocking reagents inpreventing or alleviating autoimmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases.Examples include murine experimental autoimmune encephalitis, SLE inMRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagenarthritis, diabetes mellitus in NOD mice and BB rats, and murineexperimental myasthenia gravis (see Paul ed. (1989) FundamentalImmunology, Raven Press, New York, pp. 840-856).

Upregulation of an antigen function (preferably a B lymphocyte antigenfunction), as a means of upregulating immune responses, may also beuseful in therapy. Upregulation of immune responses may be in the formof enhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response through stimulatingB lymphocyte antigen function may be useful in cases of viral infection.In addition, systemic viral diseases such as influenza, the common cold,and encephalitis might be alleviated by the administration ofstimulatory forms of B lymphocyte antigens systemically.

Alternatively, anti-viral immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide of the present invention or together with a stimulatory form ofa soluble peptide of the present invention and reintroducing the invitro activated T cells into the patient. Another method of enhancinganti-viral immune responses would be to isolate infected cells from apatient, transfect them with a nucleic acid encoding a protein of thepresent invention as described herein such that the cells express all ora portion of the protein on their surface, and reintroduce thetransfected cells into the patient. The infected cells would now becapable of delivering a costimulatory signal to, and thereby activating,T cells in vivo.

In another application, upregulation or enhancement of antigen function(preferably B lymphocyte antigen function) may be useful in theinduction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma,lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleicacid encoding at least one peptide of the present invention can beadministered to a subject to overcome tumor-specific tolerance in thesubject. If desired, the tumor cell can be transfected to express acombination of peptides. For example, tumor cells obtained from apatient can be transfected ex vivo with an expression vector directingthe expression of a peptide having B7-2-like activity alone, or inconjunction with a peptide having B7-1-like activity and/or B7-3-likeactivity. The transfected tumor cells are returned to the patient toresult in expression of the peptides on the surface of the transfectedcell. Alternatively, gene therapy techniques can be used to target atumor cell for transfection in vivo.

The presence of the peptide of the present invention having the activityof a B lymphocyte antigen(s) on the surface of the tumor cell providesthe necessary costimulation signal to T cells to induce a Tcell-mediated immune response against the transfected tumor cells. Inaddition, tumor cells which lack MHC class I or MHC class II molecules,or which fail to reexpress sufficient amounts of MHC class I or MHCclass II molecules, can be transfected with nucleic acid encoding all ora portion of (e.g., a cytoplasmic-domain truncated portion) of an MHCclass I α chain protein and β₂ microglobulin protein or an MHC class IIα chain protein and an MHC class II β chain protein to thereby expressMHC class I or MHC class II proteins on the cell surface. Expression ofthe appropriate class I or class II MHC in conjunction with a peptidehaving the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3)induces a T cell-mediated immune response against the transfected tumorcell. Optionally, a gene encoding an antisense construct that blocksexpression of an MHC class II associated protein, such as the invariantchain, can also be cotransfected with a DNA encoding a peptide havingthe activity of a B lymphocyte antigen to promote presentation of tumorassociated antigens and induce tumor specific immunity. Thus, theinduction of a T cell-mediated immune response in a human subject may besufficient to overcome tumor-specific tolerance in the subject.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include,without limitation, those described in: Current Protocols in Immunology,J. E. Coligan et al. eds., Pub. Greene Publishing Associates andWiley-Interscience (Chapter 3, In Vitro Assays for Mouse LymphocyteFunction, 3.1-3.19; Chapter 7, Immunologic Studies in Humans); Herrmannet al. (1981) Proc. Natl. Acad. Sci. USA 78:2488-92; Herrmann et al.(1982) J. Immunol. 128:1968-74; Handa et al. (1985) J. Immunol.135:1564-72; Takai et al. (1986) J. Immunol. 137:3494-500; Takai et al.(1988) J. Immunol. 140:508-12; Bowman et al. (1987) J. Virology61:1992-98; Bertagnolli et al. (1991) Cell. Immunol. 133:327-41; Brownet al. (1994) J. Immunol. 153:3079-92.

Assays for T cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins that modulate Tcell-dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski (1990) J.Immunol. 144:3028-33; and Mond and Brunswick (1991) “Assays for B cellFunction: In vitro Antibody Production” In Current Protocols inImmunology, J. E. Coligan et al. eds., Vol. 1, pp. 3.8.1-3.8.16, JohnWiley and Sons, Toronto.

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, proteins that generate predominantly Thl and CTL responses)include, without limitation, those described in: Current Protocols inImmunology, J. E. Coligan et al. eds., Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In Vitro Assays for Mouse LymphocyteFunction 3.1-3.19; Chapter 7, Immunologic Studies in Humans); Takai etal. (1986) J. Immunol. 137:3494-500; Takai et al. (1988) J. Immunol.140:508-12; Bertagnolli et al. (1992) J. Immunol. 149:3778-83.

Dendritic cell-dependent assays (which will identify, among others,proteins expressed by dendritic cells that activate naive T cells)include, without limitation, those described in: Guery et al. (1995) J.Immunol. 134:536-44; Inaba et al. (1991) J. Exp. Med. 173:549-59;Macatonia et al. (1995) J. Immunol. 154:5071-79; Porgador et al. (1995)J. Exp. Med. 182:255-60; Nair et al. (1993) J. Virol. 67:4062-69; Huanget al. (1994) Science 264:961-65; Macatonia et al. (1989) J. Exp. Med.169:1255-64; Bhardwaj et al. (1994) J. Clin. Invest. 94:797-807; Inabaet al. (1990) J. Exp. Med. 172:631-40.

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, proteins that prevent apoptosis after superantigen induction andproteins that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al. (1992) Cytometry13:795-808; Gorczyca et al. (1993) Leukemia 7:659-70; Gorczyca et al.(1993) Cancer Res. 53:1945-51; Itoh et al. (1991) Cell 66:233-43;Zacharchuk (1990) J. Immunol. 145:4037-45; Zamai et al. (1993) Cytometry14:891-97; Gorczyca et al. (1992) Internl. J. Oncol. 1:639-48.

Assays for proteins that influence early steps of T cell commitment anddevelopment include, without limitation, those described in: Antica etal. (1994) Blood 84:111-17; Fine et al. (1994) Cell. Immunol.155:111-22; Galy et al. (1995) Blood 85:2770-78; Toki et al. (1991)Proc. Nat. Acad. Sci. USA 88:7548-51.

Hematopoiesis Regulating Activity

A protein of the present invention may be useful in regulation ofhematopoiesis and, consequently, in the treatment of myeloid or lymphoidcell deficiencies. Even marginal biological activity in support ofcolony forming cells or of factor-dependent cell lines indicatesinvolvement in regulating hematopoiesis, e.g. in supporting the growthand proliferation of erythroid progenitor cells alone or in combinationwith other cytokines, thereby indicating utility, for example, intreating various anemias or for use in conjunction withirradiation/chemotherapy to stimulate the production of erythroidprecursors and/or erythroid cells; in supporting the growth andproliferation of myeloid cells such as granulocytes and monocytes /macrophages (i.e., traditional CSF activity) useful, for example, inconjunction with chemotherapy to prevent or treat consequentmyelosuppression; in supporting the growth and proliferation ofmegakaryocytes and consequently of platelets thereby allowing preventionor treatment of various platelet disorders such as thrombocytopenia, andgenerally for use in place of or complimentary to platelet transfusions;and/or in supporting the growth and proliferation of hematopoietic stemcells which are capable of maturing to any and all of theabove-mentioned hematopoietic cells and therefore find therapeuticutility in various stem cell disorders (such as those usually treatedwith transplantation, including, without limitation, aplastic anemia andparoxysmal nocturnal hemoglobinuria), as well as in repopulating thestem cell compartment post irradiation/chemotherapy, either in vivo orex vivo (i.e., in conjunction with bone marrow transplantation or withperipheral progenitor cell transplantation (homologous or heterologous))as normal cells or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify,among others, proteins that influence embryonic differentiationhematopoiesis) include, without limitation, those described in:Johansson et al. (1995) Cell. Biol. 15:141-51; Keller et al. (1993) Mol.Cell. Biol. 13:473-86; McClanahan et al. (1993) Blood 81:2903-15.

Assays for stem cell survival and differentiation (which will identify,among others, proteins that regulate lymphohematopoiesis) include,without limitation, those described in: Freshney (1994) “MethylcelluloseColony Forming Assays” In Culture of Hematopoietic Cells, Freshney etal. eds., pp. 265-68, Wiley-Liss Inc., New York; Hirayama et al. (1992)Proc. Natl. Acad. Sci. USA 89:5907-11; McNiece and Briddell (1994)“Primitive Hematopoietic Colony Forming Cells with High ProliferativePotential” In Culture of Hematopoietic Cells, Freshney et al. eds., pp.23-39, Wiley-Liss Inc., New York; Neben et al. (1994) Exp. Hematol.22:353-59; Ploemacher (1994) “Cobblestone Area Forming Cell Assay” InCulture of Hematopoietic Cells, Freshney et al. eds., pp. 1-21,Wiley-Liss Inc., New York; Spooncer et al. (1994) “Long Term Bone MarrowCultures in the Presence of Stromal Cells” In Culture of HematopoieticCells, Freshney et al. eds., pp. 163-179, Wiley-Liss Inc., New York;Sutherland (1994) “Long Term Culture Initiating Cell Assay” In Cultureof Hematopoietic Cells, Freshney et al. eds., pp. 139-162, Wiley-LissInc., New York.

Research Uses and Utilities

Polynucleotides provided by the present invention can be used by theresearch community for various purposes. The polynucleotides can be usedto express recombinant protein for analysis, characterization ortherapeutic use; as markers for tissues in which the correspondingprotein is preferentially expressed (either constitutively or at aparticular stage of tissue differentiation or development or in diseasestates); as molecular weight markers on Southern gels; as chromosomemarkers or tags (when labeled) to identify chromosomes or to map relatedgene positions; to compare with endogenous DNA sequences in patients toidentify potential genetic disorders; as probes to hybridize and thusdiscover novel, related DNA sequences; as a source of information toderive PCR primers for genetic fingerprinting; as a probe to“subtract-out” known sequences in the process of discovering other novelpolynucleotides; for selecting and making oligomers for attachment to a“gene chip” or other support, including for examination of expressionpatterns; to raise anti-protein antibodies using DNA immunizationtechniques; and as an antigen to raise anti-DNA antibodies or elicitanother immune response. Where the polynucleotide encodes a proteinwhich binds or potentially binds to another protein (such as, forexample, in a receptor-ligand interaction), the polynucleotide can alsobe used in interaction trap assays (such as, for example, that describedin Gyuris et al. (1993) Cell 75:791-803) to identify polynucleotidesencoding the other protein with which binding occurs or to identifyinhibitors of the binding interaction.

The proteins provided by the present invention can similarly be used inassays to determine biological activity, including in a panel ofmultiple proteins for high-throughput screening; to raise antibodies orto elicit another immune response; as a reagent (including the labeledreagent) in assays designed to quantitatively determine levels of theprotein (or its receptor) in biological fluids; as markers for tissuesin which the corresponding protein is preferentially expressed (eitherconstitutively or at a particular stage of tissue differentiation ordevelopment or in a disease state); and, of course, to isolatecorrelative receptors or ligands. Where the protein binds or potentiallybinds to another protein (such as, for example, in a receptor-ligandinteraction), the protein can be used to identify the other protein withwhich binding occurs or to identify inhibitors of the bindinginteraction. Proteins involved in these binding interactions can also beused to screen for peptide or small molecule inhibitors or agonists ofthe binding interaction.

Any or all of these research utilities are capable of being developedinto reagent grade or kit format for commercialization as researchproducts.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include withoutlimitation “Molecular Cloning: A Laboratory Manual” (1989) 2d ed., ColdSpring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.Maniatis, Eds., and “Methods in Enzymology: Guide to Molecular CloningTechniques” (1987) Academic Press, Berger, S. L. and A. R. Kimmel, Eds.

Nutritional Uses

Polynucleotides and proteins of the present invention can also be usedas nutritional sources or supplements. Such uses include withoutlimitation use as a protein or amino acid supplement, use as a carbonsource, use as a nitrogen source and use as a source of carbohydrate. Insuch cases the protein or polynucleotide of the invention can be addedto the feed of a particular organism or can be administered as aseparate solid or liquid preparation, such as in the form of powder,pills, solutions, suspensions or capsules. In the case ofmicroorganisms, the protein or polynucleotide of the invention can beadded to the medium in or on which the microorganism is cultured.

MU-1 proteins of the invention may also be used to immunize animals toobtain polyclonal and monoclonal antibodies which specifically reactwith the MU-1 protein and which may inhibit binding of ligands to thereceptor. Such antibodies may be obtained using the entire MU-1 as animmunogen, or by using fragments of MU-1. Smaller fragments of the MU-1may also be used to immunize animals. The peptide immunogensadditionally may contain a cysteine residue at the carboxyl terminus,and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).Additional peptide immunogens may be generated by replacing tyrosineresidues with sulfated tyrosine residues. Methods for synthesizing suchpeptides are known in the art, for example, as in Merrifield (1963) J.Amer. Chem. Soc. 85:2149-54; Krstenansky et al. (1987) FEBS Lett.211:10.

Neutralizing or nonneutralizing antibodies (preferably monoclonalantibodies) binding to MU-1 protein may also be useful therapeutics forcertain tumors and also in the treatment of conditions described above.These neutralizing monoclonal antibodies may be capable of blockingligand binding to the MU-1 receptor chain.

All patent and literature references cited herein are incorporated byreference as if fully set forth. This invention is further illustratedby the following examples, which should not be construed as limiting.

Examples Example 1 Isolation and Characterization of Murine MU-1 cDNAs

A partial fragment of the murine homolog of the MU-1 receptor wasisolated by PCR using oligonucleotides derived from the human sequence.cDNA was prepared from RNA isolated from 17-day-old murine thymus andfrom the murine 2D6 T cell line. A DNA fragment of approximately 300nucleotides was amplified from the cDNA by PCR with the followingoligonucleotides, corresponding to regions 584-603 and 876-896,respectively, of the human cDNA sequence in FIG. 1 (corresponding to SEQID NO:1):

AGCATCAAGCCGGCTCCCCC (5p) (SEQ ID NO: 11) CTCCATTCACTCCAGGTCCC (3p) (SEQID NO: 12)

Amplification was carried out using Taq polymerase in 1× Taq buffercontaining 1.5 mM of magnesium chloride for 30 cycles at 94° C. for oneminute, 50° C. for 1 minute, and 72° C. for 1 minute. The DNA sequenceof this fragment was determined, and two oligonucleotides were derivedfrom an internal portion of this fragment with the following sequences:

TTGAACGTGACTGTGGCCTT (5p) (SEQ ID NO: 13) TGAATGAAGTGCCTGGCTGA (3p) (SEQID NO: 14)The oligonucleotides were used to amplify an internal 262 nucleotidefragment of the original PCR product (corresponding to nucleotides781-1043 of the murine cDNA sequence of FIG. 1, and SEQ ID NO:9) to useas a hybridization probe to screen a cDNA library isolated from the 2D6T cell line. Filters were hybridized at 65° C. using standard 5×SSChybridization conditions and washed into SSC at 65° C. Twenty cloneswere isolated that hybridized to the probe in a screen of 426,000clones. DNA sequence was determined from two independent clones. Fulllength sequence of clone #6 confirmed that it was the full-length murinehomolog of human MU-1 (SEQ ID NO:9).

The full-length nucleotide sequence of murine MU-1 is shown in FIG. 1(corresponding to SEQ ID NO:9). The nucleotide sequence has a predictedleader sequence at nucleotides 407-463, coding sequence at nucleotides407-1993, termination codon at nucleotides 1994-1996. Nucleotides 1-406correspond to the 5′ untranslated region and nucleotides 1997-2628correspond to the 3′ untranslated region.

The predicted protein sequence of murine MU-1 is shown in FIG. 2(corresponding to SEQ ID NO:10). This murine MU-1 protein contains apredicted leader sequence determined by SPScan (score=10.1)(corresponding to amino acids 1-19 of SEQ ID NO:10), and a predictedtransmembrane domain (corresponding to amino acids 237-253 of SEQ IDNO:10). Predicted signaling motifs include the following regions: Box 1:amino acids 265-274 of SEQ ID NO:10, Box 2: amino acids 311-324 of SEQID NO:10, six tyrosine residues at positions 281, 319, 361, 369, 397,and 510 of SEQ ID NO:10. Potential STAT docking sites include: STAT 5:EDDGYPA (SEQ ID NO:18), STAT 3:YLQR (SEQ ID NO:19).

Example 2 Comparison of Human and Murine MU-1

The GAP algorithm was used to compare the human and murine MU-1 aminoacids. A comparison of the murine and human predicted protein sequencesis shown in FIG. 4. The amino acids were 65.267% identical using the GAPalgorithm. The alignment was generated by BLOSUM62 amino acidsubstitution matrix (Henikoff and Henikoff (supra)). Gap parameters=GapWeight: 8, Average Match=2.912, Length Weight=2, AverageMismatch=−2.003. Percent Similarity=69.466.

A comparison of the human and murine cDNA nucleotide sequences is shownin FIG. 3. The DNA sequences are 66.116% identical when aligned usingthe GAP algorithm. Gap Parameters: Gap Weight=50, Average Match=10.000,Length Weight=3, Average Mismatch=0.000. Percent Similarity=66.198.

Both human and mouse MU-1 proteins are members of the Type 1 cytokinereceptor superfamily. Evaluation of the sequence of both murine andhuman MU-1 reveals the presence of potential Box-1 and Box-2 signalingmotifs. Six tyrosine residues are present in the cytoplasmic domain, andcould also be important in signaling functions of MU-1. Comparison ofthe sequences of MU-1 with other members of the family suggested thepresence of potential docking sites for STAT 5 and STAT 3.

Example 3 Determination of STAT Signaling Pathways Used by Human MU-1

BAF-3 cells were engineered to express a chimeric cytokine receptorconsisting of the extracellular domain of the human EPO receptor and theintracellular domain of the MU-1 receptor. BAF-3 cells that expressedhuEPOR/MU-1(cyto) chimeric receptors proliferated in response to humansoluble EPO. These cells were analyzed to determine which STAT moleculeswere phosphorylated in response to EPO signaling. Briefly, controlunmodified parental BAF-3 cells and EPOR/MU chimeric BAF-3 cells wererested from IL-3-containing growth medium, and restimulated with eitherIL-3 or EPO for 0, 15, 30 and 60 minutes. The cells were pelleted andresuspended in ice cold lysis buffer containing orthovanadate, topreserve phosphorylated tyrosines. Equal amounts of cell lysate wereelectrophoresed by SDS-PAGE and blotted onto nitrocellulose membranesfor western analysis. Duplicate blots were stained for phosphorylatedand nonphosphorylated forms of STAT 1, 3, 5, and 6 by using antibodiesspecific for each form of the STAT molecule. HELA cells, nonactivatedand activated with alpha-interferon were used as positive controls.

These results indicated that under these specific conditions, signalingthrough MU-1 results in the phosphorylation of STAT 5 at all time pointstested (T=0, T=15′, T=30′, T=60′). Treatment of controls or the chimericBAF-3 cells with IL-3 resulted in phosphorylation of STAT 3, but notSTAT 1 or 5.

Example 4 Tissue Expression of Murine and Human MU-1 Northern Analysis

Northern blots of polyA+RNA from various tissues (Clontech, Palo Alto,Calif.) were performed as recommended by the manufacturer. For themurine blots, a 262 nucleotide fragment, corresponding to nucleotides781-1043 of FIG. 1 and SEQ ID NO:9, was used for hybridization.

A single transcript of murine MU-1 was detected in adult murine spleen,lung, and heart tissues. The larger transcript observed in human tissueswas not observed in mouse tissues.

Two transcripts of human MU-1 were detected in adult human lymphoidtissues, PBLs, thymus, spleen and lymph node, and in fetal lung.

In Situ Hybridization

In situ hybridization studies were performed by Phylogency Inc. ofColumbus, Ohio (according to the method of Lyons et al. (1990) J. Cell.Biol. 111:2427-36). Briefly, serial 5-7 micron paraffin sections weredeparaffinized, fixed, digested with proteinase K, treated withtri-ethanolamine and dehydrated. cRNAs were prepared from linearizedcDNA templates to generate antisense and sense probes. The cRNAtranscripts were synthesized according to manufacturer's conditions(Ambion) and labeled with ³⁵S-UTP. Sections were hybridized overnight,stringently washed and treated with RNase A and dipped in nuclear trackemulsion and exposed for 2-3 weeks. Control sections were hybridizedwith sense probes to indicate the background level of the procedure. Themurine probe consisted of an 186 by fragment corresponding tonucleotides 860-1064 (SEQ ID NO:9). The human probe was a 231 by PCRproduct generated from human MU-1 DNA.

Murine MU-1 expression was observed in the lymph nodes of the adultsmall intestine at germinal centers and muscularis externa. Specializedlymph nodes and Peyer's patches also exhibited murine MU-1 expression.

Human MU-1 expression was detected at germinal centers of the lymphnodules in the cortex. The medulla, which contains macrophages, wasnegative for human MU-1. In human spleen, human MU-1 expression wasdetected in the regions of white pulp but not red pulp.

Example 5 Expression of Human MU-1 in Cells and Cell Lines

RNAse protection analysis was performed on resting and activated human Tcells and the B cell lines, Raji and RPMI 8866, and the T cell lineJurkat. Human T cells were activated with anti-CD3 and anti-CD28. Thecell lines were activated by phorbol ester and ionomycin. MU-1riboprobe-producing plasmid was constructed by inserting a 231 by PCRproduct (PCR was performed by using 5′ primerCACAAAGCTTCAGTATGAGCTGCAGTACAGGAA CCGGGGA (SEQ ID NO: 15) and 3′ primerCACAGGATCCCTTTAACT CCTCTGACTGGGTCTGAAAGAT (SEQ ID NO:16)) into the BamH1and HindIII sites of pGEM3zf(−) (Promega, Madison, Wis.) vector. To makethe riboprobe, the riboprobe-producing plasmid was linearized withHindIII. The resulting DNA was phenol/chloroform extracted andprecipitated with ethanol. T7 RNA polymerase was used to make theriboprobe according to the protocol suggested by the vendor (PharMingen,San Diego, Calif.). The RNAse protection assay was performed by usingPharMingen's RiboQuant Multi-Probe Ribonuclease Protection Assay system.2.0 ug of total RNA were included in each RPA reaction; after RNAsedigestion, the protected riboprobes were run on a QuickPoint rapidnucleic acid separation system (Novex, San Diego, Calif.). Gels weredried and exposed according to the suggestion of the vendor.

Human MU-1 RNA is upregulated in anti-CD3+ anti-CD28-stimulated humanpurified CD3+ cells when compared with unstimulated populations. MU-1 isalso upregulated upon restimulation in Th and Th2-skewed T cellpopulations. The B cell lines, RPMI 8866 and Raji, constitutivelyexpress MU-1 while the Jurkat T cell line does not.

Example 6 Binding of Human MU-1 to Known Cytokines

Both human and murine Ig fusion proteins were constructed andimmobilized on Biacore chips in an effort to identify the ligand forMU-1. A variety of cell culture-conditioned media as well as a panel ofknown cytokines was evaluated for binding to MU-1. Some cytokines werealso tested in combination with other receptor chains in the family toconsider the possibility that MU-1 may require a second receptor chainfor ligand binding. The following cytokines were tested and found to benegative for MU-1 binding: mIL-2, hIL-2, hIL-15, mIL-7, TSLP, TSLP+IL-7,TSLP+IL-7R, TSLP+IL-7g, TSLP+IL-2, TSLP +IL-2 +IL-2Rbeta, IL-2Rbeta,IL-2Rgamma, IL-7R, IL-2+2Rbeta, IL-2+2Rgamma, IL-15+IL-2Rbeta,IL-15+2Rgamma, IL-7+2Rgamma, IL-2+IL-7R, IL-15+IL-7R, IL-7+IL-7R. Knownreceptors have been immobilized as well and tested for MUFc binding withnegative results. IL-15 will bind to IL-2Rb but not IL-2Rg or MUFc.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An antibody or fragment thereof that binds to a protein comprising anamino acid sequence selected from the group consisting of: (a) the aminoacid sequence of SEQ ID NO:2; (b) the amino acid sequence of SEQ ID NO:2from about amino acid 22 to about 538; (c) the amino acid sequence ofSEQ ID NO:2 from about amino acid 20 to about 538; (d) the amino acidsequence of SEQ ID NO:2 from about amino acid 22 to about 232; (e) theamino acid sequence of SEQ ID NO:2 from about amino acid 22 to about233; (f) the amino acid sequence of SEQ ID NO:2 from about amino acid 22to about 234; (g) the amino acid sequence of SEQ ID NO:2 from aboutamino acid 22 to about 235; (h) the amino acid sequence of SEQ ID NO:2from about amino acid 22 to about 236; (i) the amino acid sequence ofSEQ ID NO:2 from about amino acid 20 to about 232; (j) the amino acidsequence of SEQ ID NO:2 from about amino acid 20 to about 233; (k) theamino acid sequence of SEQ ID NO:2 from about amino acid 20 to about234; (l) the amino acid sequence of SEQ ID NO:2 from about amino acid 20to about 235; (m) the amino acid sequence of SEQ ID NO:2 from aboutamino acid 20 to about 236; (n) the amino acid sequence of SEQ ID NO:2from about amino acid 1 to about 232; (o) the amino acid sequence of SEQID NO:2 from about amino acid 1 to about 233; (p) the amino acidsequence of SEQ ID NO:2 from about amino acid 1 to about 234; (q) theamino acid sequence of SEQ ID NO:2 from about amino acid 1 to about 235;and (r) the amino acid sequence of SEQ ID NO:2 from about amino acid 1to about
 236. 2. The antibody or fragment thereof of claim 1, whereinthe amino acid sequence is SEQ ID NO:2.
 3. The antibody or fragmentthereof of claim 1, wherein the amino acid sequence is SEQ ID NO:2 fromabout amino acid 20 to about
 538. 4. The antibody or fragment thereof ofclaim 1, wherein the amino acid sequence is SEQ ID NO:2 from about aminoacid 20 to about
 235. 5. The antibody or fragment thereof of claim 1,wherein the amino acid sequence is SEQ ID NO:2 from about amino acid 20to about
 236. 6. The antibody or fragment thereof of claim 1, whereinthe amino acid sequence is SEQ ID NO:2 from about amino acid 1 to about235.
 7. The antibody or fragment thereof of claim 1, wherein the aminoacid sequence is SEQ ID NO:2 from about amino acid 1 to about
 236. 8. Anantibody or fragment thereof that binds to a protein comprising an aminoacid sequence encoded by a polynucleotide sequence selected from thegroup consisting of: (a) the nucleotide sequence of SEQ ID NO:1; (b) thenucleotide sequence of SEQ ID NO:1 from about nucleotide 236 to about1852; (c) the nucleotide sequence of SEQ ID NO:1 from about nucleotide299 to about 1852; (d) the nucleotide sequence of SEQ ID NO:1 from aboutnucleotide 293 to about 1852; (e) the nucleotide sequence of SEQ ID NO:1from about nucleotide 299 to about 931; (f) the nucleotide sequence ofSEQ ID NO:1 from about nucleotide 299 to about 934; (g) the nucleotidesequence of SEQ ID NO:1 from about nucleotide 299 to about 937; (h) thenucleotide sequence of SEQ ID NO:1 from about nucleotide 299 to about940; (i) the nucleotide sequence of SEQ ID NO:1 from about nucleotide299 to about 943; (j) the nucleotide sequence of SEQ ID NO:1 from aboutnucleotide 293 to about 931; (k) the nucleotide sequence of SEQ ID NO:1from about nucleotide 293 to about 934; (l) the nucleotide sequence ofSEQ ID NO:1 from about nucleotide 293 to about 937; (m) the nucleotidesequence of SEQ ID NO:1 from about nucleotide 293 to about 940; (n) thenucleotide sequence of SEQ ID NO:1 from about nucleotide 293 to about943; (o) a nucleotide sequence encoding the protein encoded by (b) abovein the reading frame beginning with about nucleotide 236 and varyingfrom the nucleotide sequence of (b) as a result of degeneracy of thegenetic code; (p) a nucleotide sequence encoding the protein encoded by(c) above in the reading frame beginning with about nucleotide 299 andvarying from the nucleotide sequence of (c) as a result of degeneracy ofthe genetic code; (q) a nucleotide sequence encoding the protein encodedby (d) above in the reading frame beginning with about nucleotide 293and varying from the nucleotide sequence of (d) as a result ofdegeneracy of the genetic code; (r) a nucleotide sequence encoding theprotein encoded by any one of (e)-(i) above in the reading framebeginning with about nucleotide 299 and varying from the nucleotidesequence of any one of (e)-(i) as a result of degeneracy of the geneticcode; (s) a nucleotide sequence encoding the protein encoded by any oneof (j)-(n) above in the reading frame beginning with about nucleotide293 and varying from the nucleotide sequence of any one of (j)-(n) as aresult of degeneracy of the genetic code; (t) a nucleotide sequencecapable of hybridizing under stringent conditions to the complement ofthe nucleotide sequence specified in any of (a)-(s); (u) a complement ofa nucleotide sequence capable of hybridizing under stringent conditionsto the nucleotide sequence specified in any of (a)-(s); (v) a nucleotidesequence encoding a species homologue of the sequence of SEQ ID NO:2 ora biologically active fragment thereof; and (w) an allelic variant ofthe nucleotide sequence specified in any of (a)-(n). wherein the proteincomprises an amino acid sequence of SEQ ID NO:2 or a fragment thereof.9. The antibody or fragment thereof of claim 8, wherein thepolynucleotide sequence is the nucleotide sequence of SEQ ID NO:1. 10.The antibody or fragment thereof of claim 8, wherein the polynucleotidesequence is the nucleotide sequence of SEQ ID NO:1 from about nucleotide236 to about
 1852. 11. The antibody or fragment thereof of claim 8,wherein the polynucleotide sequence is the nucleotide sequence of SEQ IDNO:1 from about nucleotide 293 to about
 1852. 12. The antibody orfragment thereof of claim 8, wherein the polynucleotide sequence is thenucleotide sequence of SEQ ID NO:1 from about nucleotide 293 to about940.
 13. The antibody or fragment thereof of claim 8, wherein thepolynucleotide sequence is the nucleotide sequence of SEQ ID NO:1 fromabout nucleotide 293 to about
 943. 14. The antibody or fragment thereofof claim 8, wherein the polynucleotide sequence is the nucleotidesequence of SEQ ID NO:1 from about nucleotide 236 to about
 940. 15. Theantibody or fragment thereof of claim 8, wherein the polynucleotidesequence is the nucleotide sequence of SEQ ID NO:1 from about nucleotide236 to about
 943. 16. A composition comprising the antibody or fragmentthereof of claim
 1. 17. A composition comprising the antibody orfragment thereof of claim
 8. 18. A method of producing an antibodycomprising inoculating an animal with a protein comprising an amino acidsequence selected from the group consisting of: (a) the amino acidsequence of SEQ ID NO:2; (b) the amino acid sequence of SEQ ID NO:2 fromabout amino acid 22 to about 538; (c) the amino acid sequence of SEQ IDNO:2 from about amino acid 20 to about 538; (d) the amino acid sequenceof SEQ ID NO:2 from about amino acid 22 to about 232; (e) the amino acidsequence of SEQ ID NO:2 from about amino acid 22 to about 233; (f) theamino acid sequence of SEQ ID NO:2 from about amino acid 22 to about234; (g) the amino acid sequence of SEQ ID NO:2 from about amino acid 22to about 235; (h) the amino acid sequence of SEQ ID NO:2 from aboutamino acid 22 to about 236; (i) the amino acid sequence of SEQ ID NO:2from about amino acid 20 to about 232; (j) the amino acid sequence ofSEQ ID NO:2 from about amino acid 20 to about 233; (k) the amino acidsequence of SEQ ID NO:2 from about amino acid 20 to about 234; (l) theamino acid sequence of SEQ ID NO:2 from about amino acid 20 to about235; (m) the amino acid sequence of SEQ ID NO:2 from about amino acid 20to about 236; (n) the amino acid sequence of SEQ ID NO:2 from aboutamino acid 1 to about 232; (o) the amino acid sequence of SEQ ID NO:2from about amino acid 1 to about 233; (p) the amino acid sequence of SEQID NO:2 from about amino acid 1 to about 234; (q) the amino acidsequence of SEQ ID NO:2 from about amino acid 1 to about 235; and (r)the amino acid sequence of SEQ ID NO:2 from about amino acid 1 to about236.
 19. A method of producing an antibody comprising inoculating ananimal with a protein comprising an amino acid sequence encoded by apolynucleotide sequence selected from the group consisting of: (a) thenucleotide sequence of SEQ ID NO:1; (b) the nucleotide sequence of SEQID NO:1 from about nucleotide 236 to about 1852; (c) the nucleotidesequence of SEQ ID NO:1 from about nucleotide 299 to about 1852; (d) thenucleotide sequence of SEQ ID NO:1 from about nucleotide 293 to about1852; (e) the nucleotide sequence of SEQ ID NO:1 from about nucleotide299 to about 931; (f) the nucleotide sequence of SEQ ID NO:1 from aboutnucleotide 299 to about 934; (g) the nucleotide sequence of SEQ ID NO:1from about nucleotide 299 to about 937; (h) the nucleotide sequence ofSEQ ID NO:1 from about nucleotide 299 to about 940; (i) the nucleotidesequence of SEQ ID NO:1 from about nucleotide 299 to about 943; (j) thenucleotide sequence of SEQ ID NO:1 from about nucleotide 293 to about931; (k) the nucleotide sequence of SEQ ID NO:1 from about nucleotide293 to about 934; (l) the nucleotide sequence of SEQ ID NO:1 from aboutnucleotide 293 to about 937; (m) the nucleotide sequence of SEQ ID NO:1from about nucleotide 293 to about 940; (n) the nucleotide sequence ofSEQ ID NO:1 from about nucleotide 293 to about 943; (o) a nucleotidesequence encoding the protein encoded by (b) above in the reading framebeginning with about nucleotide 236 and varying from the nucleotidesequence of (b) as a result of degeneracy of the genetic code; (p) anucleotide sequence encoding the protein encoded by (c) above in thereading frame beginning with about nucleotide 299 and varying from thenucleotide sequence of (c) as a result of degeneracy of the geneticcode; (q) a nucleotide sequence encoding the protein encoded by (d)above in the reading frame beginning with about nucleotide 293 andvarying from the nucleotide sequence of (d) as a result of degeneracy ofthe genetic code; (r) a nucleotide sequence encoding the protein encodedby any one of (e)-(i) above in the reading frame beginning with aboutnucleotide 299 and varying from the nucleotide sequence of any one of(e)-(i) as a result of degeneracy of the genetic code; (s) a nucleotidesequence encoding the protein encoded by any one of (j)-(n) above in thereading frame beginning with about nucleotide 293 and varying from thenucleotide sequence of any one of (j)-(n) as a result of degeneracy ofthe genetic code; (t) a nucleotide sequence capable of hybridizing understringent conditions to the complement of the nucleotide sequencespecified in any of (a)-(s); (u) a complement of a nucleotide sequencecapable of hybridizing under stringent conditions to the nucleotidesequence specified in any of (a)-(s); (v) a nucleotide sequence encodinga species homologue of the sequence of SEQ ID NO:2 or a biologicallyactive fragment thereof; and (w) an allelic variant of the nucleotidesequence specified in any of (a)-(n). wherein the protein comprises anamino acid sequence of SEQ ID NO:2 or a fragment thereof.
 20. Anantibody or fragment thereof produced by the method of claim 18, whereinthe antibody or fragment thereof binds to a polypeptide comprising theamino acid sequence of SEQ ID NO:2 or a biologically active fragmentthereof.
 21. An antibody or fragment thereof produced by the method ofclaim 19, wherein the antibody or fragment thereof binds to apolypeptide comprising the amino acid sequence of SEQ ID NO:2 or abiologically active fragment thereof.
 22. The antibody or fragmentthereof of any one of claims 1, 8, 20, and 21, wherein the antibody is amonoclonal antibody.
 23. The antibody or fragment thereof of claim 1 or8, wherein the antibody is a neutralizing antibody.
 24. The antibody orfragment thereof of claim 20 or 21, wherein the antibody is aneutralizing antibody.
 25. The antibody or fragment thereof of claim 23,wherein the antibody or fragment thereof neutralizes the activity of theprotein by blocking ligand binding to the MU-1 receptor.
 26. Theantibody or fragment thereof of claim 24, wherein the antibody orfragment thereof neutralizes the activity of the polypeptide by blockingligand binding to the MU-1 receptor.
 27. The antibody or fragmentthereof of claim 1 or 8, wherein the antibody or fragment thereofspecifically reacts with the protein.
 28. The antibody or fragmentthereof of claim 20 or 21, wherein the antibody or fragment thereofspecifically reacts with the polypeptide.